Conditional expression of TGF-β1 in skeletal muscles causes endomysial fibrosis and myofibers atrophy

Abstract

To study the effects of transforming growth factor beta 1 (TGF-β1) on fibrosis and failure of regeneration of skeletal muscles, we generated a tet-repressible muscle-specific TGF-β1 transgenic mouse in which expression of TGF-β1 is controlled by oral doxycycline. The mice developed muscle weakness and atrophy after TGF-β1 over-expression. We defined the group of mice that showed phenotype within 2 weeks as early onset (EO) and the rest as late onset (LO), which allowed us to further examine phenotypic differences between the groups. While only mice in the EO group showed significant muscle weakness, pathological changes including endomysial fibrosis and smaller myofibers were observed in both groups at two weeks after the TGF-β1 was over-expressed. In addition, the size of the myofibers and collagen accumulation were significantly different between the two groups. The amount of latent and active TGF-β1 in the muscle and circulation were significantly higher in the EO group compared to the LO or control groups. The up-regulation of the latent TGF-β1 indicated that endogenous TGF-β1 was induced by the expression of the TGF-β1 transgene. Our studies showed that the primary effects of TGF-β1 over-expression in skeletal muscles are muscle wasting and endomysial fibrosis. In addition, the severity of the pathology is associated with the total amount of TGF-β1 and the expression of endogenous TGF-β1. The findings suggest that an auto-feedback loop of TGF-β1 may contribute to the severity of phenotypes.

Figure 1. Muscle weakness caused by TGF-β1 over-expression in skeletal muscles after doxycycline removal.

(A) Expression of the TGF-β1 mRNA in various organs. TGF-β1 transcripts were detected in the two muscles, quadriceps and diaphragm, examined (lanes 1 and 2), but not in the brain, heart, lung, liver, kidney or ovary (lanes 3–7, respectively). Lane 8 is a no RT control. (B) GSM showed that muscle strength was significantly reduced in mice that developed early phenotype but not the rest of the mice over-expressing TGF-β1. The asterisks indicate significant differences with p<0.05.

Figure 2. Skeletal muscle wasting due to TGF-β1 over-expression.

(A) Skeletal muscle wasting in mice over-expressing TGF-β1, which developed muscle weakness. (B) The average muscle weight (g, mean ± s.e.m.) was reduced in these mice. “*” indicates p<0.05 and “**” indicates p<0.01.

Figure 3. TGF-β1 over-expression leads to myofiber atrophy and endomysial fibrosis.

(A) H&E and Pico-Sirius red staining showed smaller myofibers and collagen accumulation in the mice over-expressing TGF-β1. The differences were less prominent in the mice that did not develop muscle weakness at the time of muscle collection. Scale bar: 50 µm (B) Collagen deposition measurement after 2 weeks of TGF-β1 over-expression. The mice in the EO group showed significantly higher collagen deposition in comparison to LO and control mice. “*” indicates p<0.05 and “**” indicates p<0.01. (C) Fiber size distribution after 2 weeks of TGF-β1 over-expression. The myofiber size was reduced in both the EO and LO groups with more reduction in the EO group.

Figure 4. Active and latent TGF-β1 levels in serum and quadriceps after two weeks of TGF-β1 over-expression.

(A) The serum level of both active and latent TGF-β1 was significantly higher in mice in the EO group in comparison to LO and control littermates. (B) The level of both active and latent TGF-β1 in quadriceps was significantly higher in mice in the EO group in comparison to LO and control littermates. “*” indicates p<0.05 and “**” indicates p<0.01.